Akeo Hagiwara

Causal relationship between the loss of RUNX3 expression and gastric cancer.

Runx3/Pebp2alphaC null mouse gastric mucosa exhibits hyperplasias due to stimulated proliferation and suppressed apoptosis in epithelial cells, and the cells are resistant to growth-inhibitory and apoptosis-inducing action of TGF-beta, indicating that Runx3 is a major growth regulator of gastric epithelial cells. Between 45% and 60% of human gastric cancer cells do not significantly express RUNX3 due to hemizygous deletion and hypermethylation of the RUNX3 promoter region. Tumorigenicity of human gastric cancer cell lines in nude mice was inversely related to their level of RUNX3 expression, and a mutation (R122C) occurring within the conserved Runt domain abolished the tumor-suppressive effect of RUNX3, suggesting that a lack of RUNX3 function is causally related to the genesis and progression of human gastric cancer.

Tumour-amplified kinase BTAK is amplified and overexpressed in gastric cancers with possible involvement in aneuploid formation

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.

Gains, losses, and amplifications of genomic materials in primary gastric cancers analyzed by comparative genomic hybridization

By means of comparative genomic hybridization (CGH), we screened 58 primary gastric cancers for changes in copy number of DNA sequences. We detected frequent losses on 1p32–33 (21%), 3p21–23 (22%), 5q14–22 (36%), 6q16 (26%), 9p21–24 (22%), 16q (21%), 17p13 (48%), 18q11–21 (33%), and 19 (40%). Gains were most often noted at 1p36 (22%), 8p22–23 (24%), 8q23–24 (29%), 11q12–13 (24%), 16p (21%), 20p (38%), 20q (45%), Xp21–22 (38%), and Xq21–23 (43%), with high‐level amplifications at 6p21 (2%), 7q31 (10%), 8p22–23 (5%), 8q23–24 (7%), 11q13 (4%), 12p12–13 (4%), 17q21 (2%), 19q12–13 (2%), and 20q13 (2%). High‐level amplification at 8p22–23 has never been reported in any other cancer type and its frequency was as high as that reported for the MYC, MET, and KRAS genes. We narrowed down the smallest common amplicon to 8p23.1 by reverse‐painting FISH to prophase chromosomes. Southern blot analysis using one EST marker (D38736) clearly demonstrated that amplification of this exon‐like sequence had occurred in all three tumors in which amplifications at 8p22–23 had been detected by CGH. Our data provide evidence for several, previously undescribed, genomic aberrations that are characteristic of gastric cancers. Genes Chromosomes Cancer 24:299–305, 1999.

Overexpression of bax sensitizes human breast cancer MCF‐7 cells to radiation‐induced apoptosis

Resistance to apoptosis plays an important role in tumors that are refractory to chemotherapy and ionizing radiation (IR). bax, which forms a heterodimer with bcl‐2 and accelerates apoptosis, is not, or only weakly, expressed in most human breast cancer cells, and weak bax expression is considered to be related to the resistance of breast cancer cells to apoptosis. bax expression vector was introduced to human breast cancer MCF‐7 cells, which exhibit weak expression of bax, to demonstrate its role of modulating radiation‐induced apoptosis. bax overexpression in MCF‐7 cells by stable transfection does not affect viability by itself, but each stable transfectant was more sensitive to IR than the parental MCF‐7 cells. The degree of enhancement in radiosensitivity was dependent on the expression level of bax. IR upregulated p53 and p21WAFI about 5‐ to 10‐fold and downregulated bcl‐2 and bcl‐XL by 80–90% at 6 hr in both parent and bax stably transfected MCF‐7 cells to the same degree. FACS analysis and DNA electrophoresis revealed that this sensitization was due to apoptosis. We suggest that exogenous bax expression might be one of the factors determining cellular radiosensitivity in MCF‐7 breast cancer cells and may have therapeutic applications for enhancing radiation sensitivity in breast cancer cells.

Amplification and over-expression of the AIB1 nuclear receptor co-activator gene in primary gastric cancers.

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. Int. J. Cancer 89:217–223, 2000.

Differential gene expression profiles of radioresistant oesophageal cancer cell lines established by continuous fractionated irradiation

Radiation therapy is a powerful tool for the treatment of oesophageal cancer. We established radioresistant cell lines by applying fractionated irradiation in order to identify differentially expressed genes between parent and radioresistant cells. Six oesophageal cancer cell lines (TE-2, TE-5, TE-9, TE-13, KYSE170, and KYSE180) were treated with continuous 2 Gy fractionated irradiation (total dose 60 Gy). We compared expression profiles of each parent and radioresistant lines on a cDNA microarray consisting of 21168 genes. In the fractionated irradiation trial, four radioresistant sublines (TE-2R, TE-9R, TE-13R, KYSE170R) were established successfully, and we identified 19 upregulated and 28 downregulated genes common to radioresistant sublines. Upregulated genes were associated with apotosis and inflammatory response (BIRC2 and COX-2), DNA metabolism (CD73), and cell growth (PLAU). Downregulated genes were associated with apoptosis (CASP6), cell adhesion (CDH1 and CDH3), transcription (MLL3), and cell cycle (CDK6). Some of these genes were known to be associated with radiation response, such as COX-2, but others were novel. Reverse transcription–polymerase chain reaction confirmed that genes selected by cDNA microarray were overexpressed in clinical specimens of radioresistant cases. Global gene analysis of radioresistant sublines may provide new insight into mechanisms of radioresistance and effective radiation therapy.

Analysis of lymph node metastasis in early gastric cancer: Rationale of limited surgery

Since the majority of patients with early gastric cancer show long‐term survival after surgery, a special attention must be directed to preserving gastric function in these patients. Little is known about the protocol of surgical treatment appropriate for early gastric cancer patients. This study was designed to determine the appropriate surgical procedure for early gastric cancer.

Intrathoracic esophageal replacement by in situ tissue-engineered esophagus

OBJECTIVE This study aimed to evaluate in situ tissue-engineered esophagus in a canine model after experimental resection and replacement of a full circumferential defect of the intrathoracic esophagus. METHODS Two types of scaffolding were fabricated. In the KF(+) group (n = 6), oral keratinocytes and fibroblasts cultured on human amniotic membrane were sheeted on polyglycolic acid felt with smooth muscle tissue and were then rolled around tubes. In the KF(-) group (n = 6), the same procedure was followed, but the keratinocytes and fibroblasts were omitted. Both scaffolds were wrapped in omentum and implanted in the abdomen. In the KF(+) group, at 3 weeks after implantation, the scaffold developed into a tube with a well-differentiated lumen of stratified squamous cells surrounded by a thick smooth muscle-like tissue (in situ tissue-engineered esophagus). A part of the esophagus was resected and replaced by the graft in the same dogs. RESULTS In the KF(-) group, strictures developed after esophageal replacement, with almost complete obstruction within 2 to 3 weeks. In contrast, in the KF(+) group, the in situ tissue-engineered esophagus showed good distensibility and the dogs remained without feeding problems through 420 days. Esophageal peristalsis transferred food to the stomach, despite the absence of peristaltic activity in the in situ tissue-engineered esophagus itself. The thickness of the squamous epithelial layer and the smooth muscle layer of the in situ tissue-engineered esophagus were similar to that of the adjacent native esophagus. CONCLUSION The in situ tissue-engineered esophagus can successfully replace the intrathoracic esophagus, and this procedure may offer a promising surgical approach to esophageal diseases.

Chromosomal aberrations in colorectal cancers and liver metastases analyzed by comparative genomic hybridization

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%), 14q24‐32 (33.3%), 16p (40.0%), 20p (33.3%), 20q (63.3%) and 21q (36.3%), while loss was often noted at 18q12‐23 (36.7%). In metastatic tumors, there were significantly more gains and losses of DNA sequences than in primary tumors, with gains at 8q23‐24 (found in 62.5% of recurrences vs. 43.0% of primary tumors), 15q21‐26 (37.5% vs. 20.0%), 19p (43.8% vs. 20.0%) and 20q (81.3% vs. 63.3%) and losses at 18q12‐23 (50.0% vs. 36.7%). The pattern of genetic changes seen in metastatic tumors, with frequent gains at 8q23‐24 and 20q and loss at 18q12‐23, suggests the progression of colorectal cancer. We investigated a clinical follow‐up study for all patients examined by CGH and directed our attention to the genetic changes consisting of gains at 8q and 20q. The incidence of liver metastases was higher in patients with primary colorectal cancer with these genetic changes. Gains at 8q and 20q might be useful to identify patients at high risk for developing liver metastases.

Differential gene expression profiles of gastric cancer cells established from primary tumour and malignant ascites

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 up-regulated 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 γ, IL4-Stat (immune response), p27 (cell cycle) and integrin β4 (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.