Takefumi Ohga

Nuclear translocation of the Y‐box binding protein by ultraviolet irradiation

The Y‐box binding protein, YB‐1, is a member of a DNA binding protein family with a structurally and functionally conserved cold shock domain. Using Western blotting and immunohistochemical methods, larger amounts of YB‐1 were detected in the cytosol, particularly at the perinuclear region, than in the nucleus of human cancer cells. UV irradiation increased accumulation of YB‐1 in the nucleus at 20 min and thereafter. This translocation of YB‐1 into the nucleus by UV irradiation was blocked by the protein kinase inhibitor H‐7, but not HA‐1004. Both green fluorescent protein (GFP)‐YB‐1 and GFP‐YB‐1C with the C‐terminus (248–317) of YB‐1 were located mainly in the cytosol, but GFP‐YB‐1ΔC with a deletion at the C‐terminus of YB‐1 was located in the nucleus. YB‐1 is translocated into the nucleus by UV irradiation, possibly through a protein kinase C‐mediated signal transduction pathway, and the C‐terminal region of YB‐1 might be important for cytoplasmic retention of YB‐1.

EXPRESSION OF THE METASTASIS-ASSOCIATED MTA1 PROTEIN AND ITS RELATIONSHIP TO DEACETYLATION OF THE HISTONE H4 IN ESOPHAGEAL SQUAMOUS CELL CARCINOMAS

Metastasis‐associated protein MTA1 and histone deacetylase form a protein complex with histone deacetylase activity that plays an important role in histone deacetylation, alteration of chromatin structure and transcriptional control. The precise role of the MTA1 protein in the malignant progression of human cancers remains unknown, however, especially its overexpression and relationship with histone acetylation/deacetylation in experimental and clinical tumors. The expression levels of MTA1 protein and the acetylation levels of histone H4 were examined in 70 cases of surgically resected esophageal squamous cell carcinomas, using immunohistochemistry. The intensities of immunostaining of MTA1 protein and acetylated histone H4 in carcinoma tissues (Ca) were compared to normal epithelium (N) contained in the same section. Thirty of 70 cases (42.9%) displayed overexpression of MTA1 protein (N < Ca). Cancers overexpressing MTA1 protein invaded deeper into the esophageal wall (p < 0.005) and showed significantly higher degrees of lymph node metastasis (p < 0.01), higher pathological stage, more lymphatic involvement and poorer prognosis (p < 0.05) than the remaining cases. The acetylation levels of histone H4 inversely correlated to the depth of cancer invasion and pathological stage (p < 0.05), and the patients with higher level of histone H4 acetylation had a better prognosis (p < 0.05). Furthermore, immunostaining patterns of MTA1 and acetylated histone H4 were inversely correlated (p < 0.001), demonstrating the relationship of deacetylation of histone H4 in MTA1‐overexpressing carcinomas. In conclusion, the data suggest that the overexpression of MTA1 protein and acetylation level of histone H4 protein, both of which are closely related, might be useful predictors of malignant potential of esophageal squamous cell carcinomas. Thus, strategies involving inhibition of MTA1 function as well as inhibition of histone deacetylation could be novel approaches for the treatment of esophageal squamous cell carcinomas.

A novel variant of WISP1 lacking a von Willebrand type C module overexpressed in scirrhous gastric carcinoma

Scirrhous carcinoma of the stomach is characterized by rapid growth with a vast fibrous stroma, high invasiveness, and substantially a poor prognosis. Little is known of the molecular pathogenesis of this disease. Members of the emerging family of the CCN gene (for connective tissue growth factor, cysteine-rich 61, nephroblastoma overexpressed) encode cysteine-rich secreted proteins with roles in human fibrotic disorders and cancer progression. Using targeted differential displays, we identified a novel variant of the CCN family member WISP1 (Wnt-induced secreted protein 1), named WISP1v, as overexpressed in scirrhous gastric carcinomas. Predicted protein of the WISP1v completely lacks a module of Von Willebrand type C that is thought to participate in protein complex formation. Ectopic expression revealed WISP1v to be a secreted oncoprotein inducing a striking cellular transformation and rapid piling-up growth. It is noteworthy that WISP1v transfectants enhanced the invasive phenotype of co-cultured gastric carcinoma cells, while wild-type WISP1 had no such potential. These findings suggest that CCN protein WISP1v is involved in the aggressive progression of scirrhous gastric carcinoma.

Chemosensitisation of spontaneous multidrug resistance by a 1,4-dihydropyridine analogue and verapamil in human glioma cell lines overexpressing MRP or MDR1.

Multidrug resistance phenotypes in human tumours are associated with the overexpression of the 170 kDa P-glycoprotein encoded by the multidrug resistance 1 (MDR1) gene, and also with that of the non-P-glycoprotein-mediated multidrug resistance gene, MRP, which encodes a 190 kDa membrane ATP-binding protein. We have previously reported that overexpression of MRP appears to be responsible for spontaneous multidrug resistance in some human glioma cell lines (Abe et al., Int. J. Cancer, 58, 860-864, 1994). In this study, we investigated whether chemosensitising agents of P-glycoprotein-mediated multidrug resistance such as verapamil, a biscoclaurine alkaloid (cepharanthine), and a dihydropyridine analogue (NIK250) could also reverse multidrug resistance in human glioma cells. The glioma cell lines were the two MRP-expressing cell lines, T98G and IN500, an MDR1-expressing cell line, CCF-STTG1, and the MRP1 MDR1-non-expressing cell line, IN157. Verapamil and NIK250 almost completely reversed drug resistance to vincristine, etoposide and doxorubicin in T98G cells, while they also reversed drug resistance to vincristine and etoposide, but only partially to doxorubicin in IN500 cells. Cepharanthine as well as verapamil and NIK250 reversed vincristine resistance in CCF-STTG1 cells, but cepharanthine only partially reversed drug resistance in T98G and IN500 cells. The cellular accumulation of [3H]etoposide increased about 2- and 3-fold compared with control in T98G cells in the presence of verapamil and NIK250 respectively. Furthermore, the release of doxorubicin from the nuclei of T98G cells was blocked by NIK250. However, NIK250 and verapamil caused no apparent increase in vincristine accumulation in T98G cells. NIK250 or verapamil might exert inhibitory effects upon MRP function, resulting in a reversal of MRP-mediated spontaneous multidrug resistance in cultured human glioma cells.

New technique for the retraction of the liver in laparoscopic gastrectomy.

We developed a new technique for the retraction of the liver using a Penrose drain and a J-shaped retractor, which is both an easy and time-saving method that provides a good view during laparoscopic gastrectomy without damaging the liver.

p53 Mutation Profiling of Multiple Esophageal Carcinoma Using Laser Capture Microdissection to Demonstrate Field Carcinogenesis

Inactivation of the p53 tumor suppressor gene is one of the most frequent genetic alterations observed in human esophageal carcinomas. In patients with esophageal carcinoma, one of the significant pathological features of the tumor is the presence of multiple lesions within the esophagus. However, the molecular mechanisms involved in the occurrence of multiple lesions have remained elusive. To characterize p53 alterations in multiple esophageal carcinomas and to study their roles in carcinogenesis, we performed p53 immunohistochemical and p53 mutation analyses using laser capture microdissection on surgically resected human esophageal carcinomas from 11 patients: 9 patients with multiple esophageal carcinomas, 1 with an intramural metastasis lesion within the esophagus and 1 with an intraepithelial carcinoma lesion contiguous to the main lesion. In each of the patients with multiple esophageal carcinomas, we examined samples from 1 main lesion and 1 representative concomitant lesion. Molecular analyses of samples from fresh‐frozen normal tissues and tumor tissues of the main lesion (whole tumor) were also performed by the same method. p53 protein accumulation was observed in 16 (72.7%) of 22 lesions from the 11 cases. No p53 mutation was found in normal esophageal tissues. In the 9 cases of multiple esophageal carcinomas, point mutations were detected in the whole tumor in 1 (11.1%) case, in the microdissected tumor samples of main lesions in 3 (33.3%) cases and in the microdissected tumor samples of concomitant lesions in 3 (33.3%) cases. For the microdissected tumor samples, there was a 54.5% (12/22) concordance rate between the results of immunostaining and molecular analysis. In the 8 cases of whole tumors in which a p53 mutation was not observed, 2 cases revealed p53 mutation in the microdissected tumor samples of the main lesion. All 6 cases of multiple esophageal carcinomas that showed a p53 mutation in the microdissected tumor sample had a discordant p53 mutational status between the main and concomitant lesions. In contrast, both the intramural metastasis lesion and the intraepithelial carcinoma contiguous to the main lesion showed p53 mutational patterns identical to those of the main lesions. In conclusion, the analysis of microdissected DNA by laser capture microdissection is useful for characterizing the heterogeneity of the p53 gene mutation in multiple carcinoma lesions that cannot be accurately analyzed in whole esophageal tumor DNA. The finding of different p53 gene mutations among multiple esophageal carcinoma lesions suggest further evidence of multicentric or field carcinogenesis of the human esophagus.

Interrelation Between Expression of Matrix Metalloproteinase 7 and β-Catenin in Esophageal Cancer

Matrix metalloproteinase 7 (MMP-7) may play a key role in the progression of various human malignant tumors. Nuclear β-catenin enhances the activating expression of MMP-7 genes by binding with the T-cell factor/lymphoid enhancer factor family of transcription factors. We immunohistochemically examined the expression of MMP-7 and β-catenin to better understand the significance of these factors in the progression of esophageal squamous cell carcinoma. The entire coding region of β-catenin exon 3 was also analyzed by direct sequencing in all cases. We found that MMP-7 was expressed in 7 (20.6%) of 34 esophageal squamous cell carcinomas. There was a significant relationship between MMP-7 expression and tumor invasion into adjacent structures (P < 0.05). Aberrant nuclear expression of β-catenin was found in 12 of 34 (35.3%) esophageal cancers and correlated with MMP-7 expression, the statistical difference being (P < 0.05). None of the 34 esophageal cancers examined carried mutations in β-catenin exon 3. MMP-7 expression correlates with penetrating tumor progression in esophageal cancer. Nuclear translocation of β-catenin, without mutations in β-catenin exon 3, is associated with MMP-7 expression.

Genomic organization of the human Y-box protein (YB-1) gene

The human Y-box protein (YB-1) is a member of a family of DNA-binding proteins containing a highly conserved cold shock domain. The genomic organization of the human YB-1 gene was determined from five overlapping genomic clones that encompassed all exons of the gene. Sequence analysis of these clones revealed that human YB-1 spans approximately 19 kb of genomic DNA and contains eight exons. The cold shock domain is encoded by exons 1-5. Both exon-splitting and codon-splitting in the region of the gene encoding the cold shock domain are similar to those in the corresponding region of another Y-box binding protein, dbpA. Exon-intron structures and nucleotide sequences of the regions encoding the N-terminal and C-terminal domains of the two proteins differ markedly between YB-1 and dbpA. These observations suggest that YB-1 and dbpA arose by duplication of a common ancestral gene encoding all these domains.

Esophageal replacement by colon interposition with microvascular surgery for patients with thoracic esophageal cancer: The utility of superdrainage

Replacing the thoracic esophagus with the colon is one mode of reconstruction after esophagectomy for esophageal cancer. There is, however, a high incidence of postoperative necrosis of the transposed colon. This study evaluated the outcomes of colon interposition with the routine use of superdrainage by microvascular surgery. Twenty-one patients underwent colon interposition from 2004 to 2009. The strategy for colon interposition was to: (i) use the right hemicolon; (ii) reconstruct via the subcutaneous route; (iii) perform a microvascular venous anastomosis for all patients; and (iv) perform a microvascular arterial anastomosis when the arterial blood flow was insufficient. The clinicopathologic features, surgical findings, and outcomes were investigated. The colon was used because of a previous gastrectomy in 18 patients (85.7%) and synchronous gastric cancer in three patients (14.3%). Eight patients (38.1%) underwent preoperative chemoradiotherapy including three (14.3%) treated with definitive chemoradiotherapy. Seven patients (33.3%) underwent microvascular arterial anastomosis to supplement the right colon blood supply. Pneumonia occurred in four patients (19.0%). Anastomotic leakage was observed in five patients (23.8%); however, no colon necrosis was observed. The 3-year and 5-year overall survival rates were both 50.6%. Colon interposition with superdrainage results in successful treatment outcomes. This technique is one option for colon interposition employing the right hemicolon.

Clinical Characteristics of Gastric Cancer with Metastasis to the Lymph Node along the Superior Mesenteric Vein (14v)

Aim: We investigated the clinical significance of metastasis to the lymph node (LN) along the superior mesenteric vein (14v) in gastric cancer. Methods: A retrospective study of 2,513 gastrectomy patients with a 14v dissection was done using the Ganken Igan Database. Results: The incidence of 14v metastasis correlated with tumor location, depth of tumor invasion, regional LN metastases, peritoneal metastasis, peritoneal cytology-positive, hepatic metastasis and postoperative recurrence (p < 0.01). Metastases to the infrapyloric LN (6), suprapyloric LN (5) and left paracardial LN (2) were independent variables affecting 14v metastasis (p < 0.05), and the 6 status was a useful predictive factor for a 14v-negative status with a low false-negative rate (1.9%). The patients with 14v metastasis after curative surgery demonstrated a significantly lower survival rate than those without (5-year overall survival rate; 11.3 vs. 60.2%, p < 0.0001). In them, LN around the abdominal aorta (16)-positive group showed a significantly lower survival rate than the negative group (p < 0.05). Conclusions: Advanced gastric cancer with invasion to the lower stomach often metastasizes to 14v, and the 6 status can predict 14v negative. Most patients with 14v metastasis have a poor prognosis, similar to those with systemic metastasis, although some such patients may benefit from a curative dissection.