Sakakura C

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.

RUNX3 Suppresses Gastric Epithelial Cell Growth by Inducing p21WAF1/Cip1 Expression in Cooperation with Transforming Growth Factor β-Activated SMAD

ABSTRACT RUNX3 has been suggested to be a tumor suppressor of gastric cancer. The gastric mucosa of the Runx3-null mouse develops hyperplasia due to enhanced proliferation and suppressed apoptosis accompanied by a decreased sensitivity to transforming growth factor β1 (TGF-β1). It is known that TGF-β1 induces cell growth arrest by activating CDKN1A (p21 WAF1 /Cip1 ), which encodes a cyclin-dependent kinase inhibitor, and this signaling cascade is considered to be a tumor suppressor pathway. However, the lineage-specific transcription factor that cooperates with SMADs to induce p21 expression is not known. Here we show that RUNX3 is required for the TGF-β-dependent induction of p21 expression in stomach epithelial cells. Overexpression of RUNX3 potentiates TGF-β-dependent endogenous p21 induction. In cooperation with SMADs, RUNX3 synergistically activates the p21 promoter. In contrast, RUNX3-R122C, a mutation identified in a gastric cancer patient, abolished the ability to activate the p21 promoter or cooperate with SMADs. Furthermore, areas in mouse and human gastric epithelium where RUNX3 is expressed coincided with those where p21 is expressed. Our results suggest that at least part of the tumor suppressor activity of RUNX3 is associated with its ability to induce p21 expression.

Prophylaxis with carbon-adsorbed mitomycin against peritoneal recurrence of gastric cancer

Attempts to prevent peritoneal carcinomatosis after surgery for gastric cancer by intraperitoneal administration of anticancer drugs have not been successful, largely because the drugs are not retained in the peritoneal cavity. We have assessed the prophylactic efficacy of a delayed-release preparation--mitomycin adsorbed onto activated charcoal (M-CH). 50 patients with gastric cancer and serosal infiltration were randomly assigned intraperitoneal treatment with M-CH (50 mg mitomycin intraoperatively) or no anticancer prophylaxis (control). Survival rates for the 3 years of follow-up were significantly higher among the 24 M-CH recipients (1 was lost to follow-up) than among the 25 controls (p less than 0.01). There were significant differences in survival between the groups at 1.5 years after randomisation (difference 34.6% [95% confidence interval 8.5-60.8%]; p less than 0.01) and at 2.0, 2.5, and 3.0 years (41.7% [14.2-69.1%]; p less than 0.005). The concentration of mitomycin was significantly higher in peritoneal exudate than in plasma for 24 h after drug administration. Side-effects were slight and well tolerated. Thus, peroperative intraperitoneal treatment with M-CH seems to improve survival after gastrectomy for gastric cancer, presumably by a prophylactic effect on peritoneal recurrence.

Relation between cyclooxygenase‐2 expression and tumor invasiveness and patient survival in transitional cell carcinoma of the urinary bladder

Expression of the inducible form of cyclooxygenase (COX)‐2 is known to correlate with development of transitional cell carcinoma (TCC) of the human urinary bladder. However, the clinical significance of COX‐2 expression with respect to clinicopathologic findings and patient survival is unknown.

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.

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.

Chromosomal aberrations in human hepatocellular carcinomas associated with hepatitis C virus infection detected by comparative genomic hybridization

SummaryThirty-five hepatocellular carcinomas (HCCs) associated with hepatitis C virus (HCV) were analysed by comparative genomic hybridization (CGH), to screen for changes in copy-number of DNA sequences. Chromosomal losses were noted in 1p34–36 (37%), 4q12–21 (48%), 5q13–21 (35%), 6q13–16 (23%), 8p21–23 (28%), 13q (20%), 16q (33%) and 17p13 (37%). Gains were noted in 1q (46%), 6p (20%), 8q21–24 (31%) and 17q (43%). High level gains indicative of gene amplifications were found in 7q31 (3%), 11q13 (3%), 14q12 (6%) and 17q12 (3%); amplification at 14q12 may be characteristic for HCCs. No significant difference in chromosomal aberrations was noted between carcinomas associated with HCV-infection in our study and those reported earlier in HCCs infected with hepatitis B virus (HBV), indicating that both HBV- and HCV-related carcinomas may progress through a similar cascade of molecular events.

Sphingosine induces apoptosis in androgen‐independent human prostatic carcinoma DU‐145 cells by suppression of bcl‐XL gene expression

Our recent studies have suggested that sphingosine, an endogenous protein kinase C (PKC) inhibitor, may mediate apoptosis induced by a phorbol ester (PMA) in human promyelocytic leukemia HL‐60 cells [Ohta et al. Cancer Res. 1995;55:691–697], and that the apoptotic induction by both PMA and sphingosine is accompanied by down‐regulation of bcl‐2, a gene which acts to prevent apoptotic cell death [Sakakura et al. FEBS Lett. 1996;397:177–180]. In this study, we examined the sphingosine‐induced apoptosis of the androgen‐independent human prostatic carcinoma cell line DU‐145, which expresses bcl‐XL and Bax but not bcl‐2, and found that treatment of DU‐145 cells with sphingosine suppressed bcl‐XL in both mRNA and protein levels but did not change bax expression at all. In contrast, in apoptotic cells treated with a PKC inhibitor, staurosporine, no effect on bcl‐XL or bax expression was observed. The initial metabolites of sphingosine in the cells, ceramide and sphingosine 1‐phosphate, failed to induce apoptosis. These results indicate that, in DU‐145 cells, sphingosine, but not its metabolites, induces apoptosis through down‐regulation of bcl‐XL, independently of PKC inhibition. Our present results, together with previous observations, strongly suggest that apoptosis regulatory genes differ according to cell type and apoptosis induction through sphingosine is accompanied by inhibition of either bcl‐2 or bcl‐XL activity in these cells.

Regeneration of skeletal muscle using in situ tissue engineering on an acellular collagen sponge scaffold in a rabbit model.

Because of the limited ability of skeletal muscle to regenerate, resection of a large amount of muscle mass often results in incomplete recovery due to nonfunctional scar tissue. The aim of this study was to regenerate skeletal muscle using in situ tissue engineering in a rabbit model. In 18 male rabbits, a muscle defect (1.0 × ∼1.0 × ∼0.5 cm) was created in the vastus lateralis of both legs. A piece of cross-linked atelocollagen sponge was then inserted into the defect in one leg, whereas the defect in the other leg was left untreated. Both defects were finally covered with fascia. Twenty-four weeks after surgery, the defect that had been filled with the cross-linked atelocollagen sponge scaffold showed mild concavity and slight adhesion to the fascia, while the control side showed severe scar formation and shrinkage. Histologically, the regenerating myofibers at the site containing the collagen sponge were greater in number, diameter, and length than those at the control site. These results indicate that cross-linked atelocollagen sponge has the potential to act as a scaffold for muscle tissue regeneration.