Summary Our recent analysis of gastric cancers using comparative genomic hybridization (CGH) revealed a novel high frequent copy number increase in the long arm of chromosome 20. Tumour-amplified kinase BTAK was recently cloned from breast cancers and mapped on 20q13 as a target gene for this amplification in human breast cancers. In the study presented here, we analysed BTAK copy-number and expression, and their relation to the ploidy pattern in 72 primary gastric cancers. Furthermore, wild-type BTAK and its deletion mutants were transfected to gastric cancers to examine changes in cell proliferation and DNA ploidy pattern. Evaluation of 72 unselected primary gastric cancers found BTAK amplification in 5% and overexpression in more than 50%. All four clinical samples with BTAK amplification showed aneuploidy and poor prognosis. Transfection of BTAK in near-diploid gastric cancers induced another aneuploid cell population. In contrast, the c-terminal-deleted mutant of BTAK induced no effect in DNA ploidy pattern and inhibited gastric cancer cell proliferation. These results suggest that BTAK may be involved in gastric cancer cell aneuploid formation, and is a candidate gene for the increase in the number of copies of the 20q, and thus may contribute to an increase in the malignant phenotype of gastric cancer.

Our analysis of chromosomal aberrations in primary gastric cancers using comparative genomic hybridization has revealed novel, high and frequent copy number increases in the long arm of chromosome 20, indicating that this region contains novel amplified genes involved in gastric cancer progression. AIB1, a member of the steroid receptor co-activator-1 family, has been cloned on 20q12 as a candidate target gene for this amplification in human breast cancers. In this study, we examined the numbers of AIB1 copies as well as their expression and relation to clinico-pathological features in 72 primary gastric cancers. AIB1 amplification was observed in 7% and over-expression in 40% of the specimens. AIB1 amplification always coincided with its over-expression, but several cases showed AIB1 over-expression without amplification, suggesting that expression of AIB1 is regulated not only by gene amplification but also by other mechanisms, such as transcriptional activation, in human gastric cancer. Gastric cancers with AIB1 amplification showed extensive lymph node metastases, liver metastases and poorer prognosis compared to those without amplification. Our results suggest that amplification and over-expression of AIB1 are likely to increase the number of malignant phenotypes of gastric cancers and that it can be expected to be useful as a marker of poor prognosis.

Advanced gastric cancer is often accompanied by metastasis to the peritoneum, resulting in a high mortality rate. Mechanisms involved in gastric cancer metastasis have not been fully clarified because metastasis involves multiple steps and requires a combination of altered expressions of many different genes. Thus, independent analysis of any single gene would be insufficient to understand all of the aspects of gastric cancer peritoneal dissemination. In this study, we performed a global analysis of the differential gene expression of a gastric cancer cell line established from a primary main tumour (SNU-1) and of other cell lines established from the metastasis to the peritoneal cavity (SNU-5, SNU-16, SNU-620, KATO-III and GT3TKB). The application of a high-density cDNA microarray method made it possible to analyse the expression of approximately 21 168 genes. Our examinations of SNU-5, SNU-16, SNU-620, KATO-III and GT3TKB showed that 24 genes were upregulated and 17 genes down-regulated besides expression sequence tags. The analysis revealed the following altered expression such as: (a) up-regulation of CD44 (cell adhesion), keratins 7, 8, and 14 (epitherial marker), aldehyde dehydrogenase (drug metabolism), CD9 and IP3 receptor type3 (signal transduction); (b) down-regulation of IL2 receptor g, IL4-Stat (immune response), p27 (cell cycle) and integrin b4 (adhesion) in gastric cancer cells from malignant ascites. We then analysed eight gastric cancer cell lines with Northern blot and observed preferential up-regulation and down-regulation of these selected genes in cells prone to peritoneal dissemination. Reverse transcriptase -polymerase chain reaction confirmed that several genes selected by DNA microarray were also overexpressed in clinical samples of malignant ascites. It is therefore considered that these genes may be related to the peritoneal dissemination of gastric cancers. The results of this global gene expression analysis of gastric cancer cells with peritoneal dissemination, promise to provide a new insight into the study of human gastric cancer peritoneal dissemination.

Comparative genomic hybridization (CGH) was used to screen for changes in the number of DNA sequence copies in 30 primary colorectal cancers and 16 liver metastases, to identify regions that contain genes important for the development and progression of colorectal cancer. In primary colorectal cancer, we found frequent gains at 7p21 (36.7%), 7q31-36 (30%), 8q23-24 (43.0%), 12p (30%) Many detailed reports have been published on the carcinogenesis of colorectal cancer. Several genetic aberrations are required for tumor initiation and progression. 1 These aberrations include activation of the SRC and RAS oncogenes 2,3 along with inactivation of the FAP 4,5 and DCC 6 tumor suppressors and loss of TP53 function. 7 However, genetic changes involved in the progression and metastases of colorectal cancer remain unclear. To investigate differences in chromosomal aberrations between primary colorectal cancer and metastatic liver tumor, we used comparative genomic hybridization (CGH). CGH is based on dual-color fluorescence in situ hybridization (FISH) using differentially labeled tumors and reference DNA as a probe. 8,9 The efficacy of CGH is based on the concept that regions with an increase in the number of copies reveal sites that may contain dominantly acting oncogenes, whereas regions with a reduction may harbor putative tumorsuppressor genes. 8 Our aim was to generate a comprehensive picture of genomic and chromosomal aberrations that occur during the development of metastases of colorectal cancers and to identify those chromosomal regions that contain genes important for the development and progression of colorectal cancer.,Colorectal adenoma and adenocarcinoma have previously been analyzed by CGH, 10 as have primary and metastatic liver tumors. 11,12 These results supported ours; above all, we emphasize that some important genes appear to be contained in 20q and to play an important role in liver metastases. Furthermore, we examined the follow-up surveys of all cases and compared cases with and without loss at 18q and with gains at 8q and 20q to determine the usefulness of these genetic alterations as prognostic markers. MATERIAL AND METHODS Samples and DNA preparationPrimary tumors and metastatic liver tumors from 44 cancer patients with colorectal cancer (Table I) were classified according to the UICC system. These samples were provided by the Department of Digestive Surgery, Kyoto Prefectural University of Medicine (Kyoto, Japan). After surgical resection, tissues were frozen and stored at -80°C and DNA was extracted. CGHCGH was performed as described previously. 13 In brief, tumor DNA was labeled with FITC-dUTP and controlled DNA and extracted from a normal male with the aid of Texas red-dUTP using nick translation. Metaphase spreads were denatured at 75°C for 2.1 min in a formamide solution [70% formamide, 2 ϫ SCC (pH 7.0)] and dehydrated in an ethanol series of 70%, 85% and 100%. Labeled tumor and normal DNAs together with 10 g unlabeled cot-1 DNA was mixed with 50% formamide, 10% dextran sulfate and 2 ϫ SSC...

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