Our previous comparative genomic hybridization (CGH) study revealed a novel amplified region at 15q26 in two cell lines established from diffuse types of gastric cancer (GC). In this amplified region, FES and IGF1R, known targets on 15q26, were located telomeric to the amplicon in the two cell lines, HSC39 and 40A, suggesting that another tumor-associated gene exists in this region. While screening expressed sequence tags (ESTs) for novel genes in this region, we identified the IQGAP1 amplification. IQGAP1 has been reported to encode a ras GAP-related protein, and its interaction with cadherin and/or -catenin induces a dissociation of -catenin from the cadherin-catenin complex, one of the mechanisms for cell-cell adhesion. Northern and Western blot analyses revealed that amplification of this gene was accompanied by corresponding increases in mRNA and protein expression. Moreover, immunocytochemical staining showed that overexpressed IQGAP1 accumulated at the membrane, suggesting its colocalization with -catenin. Taken together, these findings suggest that IQGAP1 may be one of the target genes in the 15q26 amplicon correlated with a malignant phenotype of gastric cancer cells, such as diffuse and invasive characteristics, through the disruption of E-cadherin-mediated cell-cell adhesion.

408SHORT COMMUNICATION indicating the presence of one or more tumor suppressor genes in the region (White et al. 1995). Since hCAD acts as a key enzyme for the cleavage of DNA during apoptotic processes, it is a likely candidate for one of these tumor suppressors, because its inactivation may cause failure of cells to undergo apoptotic DNA degradation . As a step toward exploring somatic mutations of the hCAD in various types of malignant tumors, we determined its genomic structure and identified single-nucleotide polymorphisms (SNPs) within two exons, as well as a highly polymorphic dinucleotide repeat at the hCAD locus. Results and discussionWe used a full-length cDNA clone (4.2 kb), designated pKT-hCAD (Mukae et al. 1998), to screen a cosmid library prepared from human peripheral lymphocytes. Two cosmids containing genomic fragments of the hCAD were isolated. Hybridization of these cosmids with a (GT) 10 probe on Southern blots, after digestion by RsaI, detected a CA-repeat in one of the fragments. The subcloned fragment was sequenced, to reveal a (CT)m(CA)n microsatellite within the 5Ј region of hCAD.The polymerase chain reaction (PCR) primers designed to flank this repeat sequence for the analysis of polymorphism were: forward, 5Ј-CAAGCTAACTCAGTTGCA-TG; reverse, 5Ј-GCATGGACTGTGTCCTTGAC. PCR experiments were performed in volumes of 10µl containing 10ng genomic DNA, 0.25 units of EX-Taq DNA polymerase (Takara, Tokyo, Japan), 1ϫ PCR buffer (67 mM Tris [pH 8.8], 16.6 mM NH 2 SO 4 , 6.7 µM ethylenediaminetetraacetic acid (EDTA), 10 mM β-mercaptoethanol), 250 µM dNTPs, and 1 pmol each of α[32 P] end-labeled forward primer and non-labeled reverse primer. Cycle conditions were 94°C for 2 min, then 35 cycles of 94°C for 30 s, 62°C for 30 s, and 72°C for 30 s, with final extension for 5 min at 72°C. The PCR products were electrophoresed in 0.3-mm-thick denaturing 5% polyacrylamide gels containing 30% formamide and 7.7 M urea, at 2,200 V for 2-4h. The Abstract Caspase-activated DNase (CAD) cleaves chromosomal DNA during apoptosis. We determined its genomic structure and identified single-nucleotide polymorphisms (SNPs) within exons 5 and 7, as well as a highly polymorphic dinucleotide repeat of (CT)m(CA)n within the 5Ј region of the human CAD gene (hCAD). The genomic structure of hCAD presented here, together with information concerning SNPs within the gene, as well as a highly polymorphic (CT)m(CA)n repeat fragment at the hCAD locus, may assist in the construction of genetic maps for exploring gene(s) that play pivotal roles in carcinogenesis.