Satoshi Miyake

CDX2, a homeobox transcription factor, upregulates transcription of the p21/WAF1/CIP1 gene.

The CDX2 homeobox transcription factor plays key roles in intestinal development and homeostasis. CDX2 is downregulated during colorectal carcinogenesis, whereas overexpression of CDX2 results in growth inhibition and differentiation of colon carcinoma and intestinal cells. However, the means by which CDX2 functions remain poorly understood. p21/WAF1/CIP1 is one of the cyclin-dependent kinase inhibitors. In addition to its role in cell cycle control, p21 plays critical roles in differentiation and tumor suppression. The overlapping in both the expression and function of CDX2 and p21 in the small intestine and colon strongly suggests a link between these two genes. By means of luciferase reporter and electrophoretic mobility shift assays, we show here that CDX2 transactivated and physically interacted with the promoter of p21 in a p53-independent manner. Moreover, overexpression of CDX2 increased the mRNA expression of p21 in HT-29 colon carcinoma cells, as demonstrated by reverse transcription–polymerase chain reaction. These data suggest that p21 is a transcriptional target of CDX2. Our results may thus provide a new mechanism underlying the functions of CDX2.

PDX1 homeobox protein expression in pseudopyloric glands and gastric carcinomas

Background and aims: Although it has been reported that intestinal metaplasia implicated in gastric carcinogenesis is induced by the ParaHox gene CDX2, it is unclear which genes are responsible for the formation of pseudopyloric glands and whether they play a role in gastric carcinogenesis. Pancreatic-duodenal homeobox 1 (PDX1) is also a ParaHox gene which contributes to the genesis and development of the pancreas, duodenum, and antrum. To clarify its significance for the formation of pseudopyloric glands and gastric carcinogenesis, we investigated expression of PDX1 and mucin in gastric carcinomas and surrounding mucosa. Methods: Gastric carcinoma tissues from 95 patients were used for immunohistochemical analyses of PDX1, and mucins MUC6 and MUC5AC. Results: PDX1 was found to be frequently expressed in pseudopyloric glands and intestinal metaplasia. MUC6 was more abundant than MUC5AC in pseudopyloric glands while higher levels of MUC5AC than MUC6 were evident in intestinal metaplasia. The frequency of PDX1 positive reactivity was higher in differentiated type carcinomas (39/43, 90.7%) and T1 carcinomas (42/43, 97.7%) than in undifferentiated type (33/52, 63.5%) and T2–4 (30/52, 57.7%) carcinomas. PDX1 and MUC6 double positive expression was observed in carcinomas, respectively, including the corpus, and also correlated with histological type and depth of invasion. In contrast, no link was apparent between PDX1 and MUC5AC double positive reactivity and histological type. Conclusion: Our study suggests that PDX1 plays an important role in the development of pseudopyloric glands, and that pseudopyloric glands may reflect a condition associated with gastric carcinogenesis.

Transcriptional regulation of the human DNA methyltransferase 3A and 3B genes by Sp3 and Sp1 zinc finger proteins

The DNMT3A (DNA methyltransferase 3A) and DNMT3B genes encode putative de novo methyltransferases and show complex transcriptional regulation in the presence of three and two different promoters respectively. All promoters of DNMT3A and DNMT3B lack typical TATA sequences adjacent to their transcription start sites and contain several Sp1-binding sites. The importance of these Sp1-binding sites was demonstrated by using a GC-rich DNA-binding protein inhibitor, mithramycin A, i.e. on the basis of decrease in the promoter activities and mRNA expression levels of DNMT3A and DNMT3B. Overexpression of Sp1 and Sp3 up-regulated the promoter activities of these two genes. The physical binding of Sp1 and Sp3 to DNMT3A and DNMT3B promoters was confirmed by a gel shift assay. Interestingly, Sp3 overexpression in HEK-293T cells (human embryonic kidney 293T cells) resulted in 3.3- and 4.0-fold increase in DNMT3A and DNMT3B mRNA expression levels respectively by quantitative reverse transcriptase-PCR, whereas Sp1 overexpression did not. Furthermore, an antisense oligonucleotide to Sp3 significantly decreased the mRNA levels of DNMT3A and DNMT3B. These results indicate the functional importance of Sp proteins, particularly Sp3, in the regulation of DNMT3A and DNMT3B gene expression.

Evaluation of an Indicator for Lymph Node Metastasis of Esophageal Squamous Cell Carcinoma Invading the Submucosal Layer

Lymph node metastasis is a major prognostic factor for esophageal squamous cell carcinoma (ESCC). In recent years, endoscopic mucosal resection (EMR) has been developed with excellent results for the treatment of the superficial ESCC. To make the EMR treatment successful, it is important to establish a good indicator to identify ESCC patients at a high risk of lymph node metastasis. In this study, we examined clinicopathological and immunohistochemical factors to investigate the factors involved in lymph node metastasis of ESCC invading to the submucosal layer (sm‐ESCC). Surgical specimens from 84 sm‐ESCC patients were examined. Among 84 sm‐ESCC patients, 33 (39.3%) had lymph node metastases. Clinicopathologically, tumor depth, lymphatic invasion and blood vessel invasion showed significant correlations with lymph node metastasis by univariate analysis. Tumor depth and lymphatic invasion showed significant correlations by multivariate analysis of these factors. Immunohistochemically, P53 accumulation was observed in 45 cases (53.6%), cyclin D1 overexpression in 25 (29.8%), and pRB in 65 (77.4%). P53 accumulation, cyclin D1 overexpression and MIB‐1 Labeling Index were significantly associated with lymph node metastasis by univariate analysis, and P53 accumulation showed a significant correlation with lymph node metastasis by multivariate analysis. Among tumor depth, lymphatic invasion and P53 accumulation, tumor depth and lymphatic invasion were significantly correlated with lymph node metastasis (P=0.0023 and P=0.0092, respectively) by multivariate analysis. These data suggest that tumor depth and lymphatic invasion can be considered as good indicators for lymph node metastasis among patients with sm‐ESCC. In addition, P53 accumulation could be helpful to identify the patients who need additional treatment after EMR.

Transcription factor GATA-5 selectively up-regulates mucin gene expression

Purpose Overlapping expression patterns of epithelial mucins, MUC1-MUC6, and transcription factor GATA-5 were reported previously. However, the functional relationship between them is poorly understood. The aim of the current study is to elucidate whether or not expression of mucin genes is regulated by GATA-5.Methods GATA-5 was transiently overexpressed in COS-7 and 293T cells by plasmid transfection and/or adenovirus infection. GATA-5 expression was confirmed by Western blot analysis. Expression of mucin genes was studied by reverse-transcription-PCR. Reporter gene assays were employed to analyze the effect of GATA-5 on the promoter activity of mucin genes.Results mRNA levels of MUC2, MUC3, and MUC4 were increased, whereas those of MUC1, MUC5AC, MUC5B, and MUC6 remained unchanged upon the overexpression of GATA-5 in both COS-7 and 293T cells. By means of luciferase assay, GATA-5 was found to activate the promoters of the human MUC2 and MUC4 genes. GATA-5 lacking the zinc finger domain impaired these functions.Conclusions These findings indicate that GATA-5 may play important roles in the regulation of mucin expression and gastrointestinal epithelial cell differentiation.

A new technique for endoscopic esophageal mucosectomy using a transparent overtube with intraluminal negative pressure (np-EEM)

Abstract: We devised a new technique for endoscopic esophageal mucosectomy using a transparent overtube (OT) with intraluminal negative pressure (np‐EEM) (Figs. 1,2). Basic studies were performed on six dogs. The following two methods were utilized in the np‐EEM technique in line with standard approaches used for mucosal resection: (1) A “snare” method was used in which the esophageal mucosa protruded, similar to a polyp, through the slit of the OT and was resected by an electro‐snare passing through the endoscope biopsy channel or the injection channel of the OT. A high‐frequency current was used for the resection (Figs. 2‐a 2‐b, Fig. 3, Color). (2) A “cutter” method was used in which the mucosal protrusion was resected by a recently developed electro‐cutter (Figs. 2‐a, 2‐c, 2‐d, Fig. 3, Color).

Clonal Heterogeneity in Human Esophageal Squamous Cell Carcinomas on DNA Analysis

Cancers are thought to arise through multistep accumulation of somatic mutations in the progeny of a single cell. Multiple mutations may induce molecular intratumor heterogeneity. Therefore, we examined molecular clonal heterogeneity in esophageal squamous cell carcinomas. Twenty‐four esophageal squamous cell carcinomas and associated lymph node metastases were examined for microsatellite alterations, and abnormalities of the p53 and transforming growth factor‐β type II receptor (TGF‐β RII) genes. There were eight cases (33%) showing different patterns of loss of heterozygosity in primary tumors and metastatic lymph nodes with microsatellite markers. On the other hand, the abnormalities of p53 were identical in all these cases. No mutation was detected in the simple repeated sequences of the TGF‐β RII gene. These results indicate that molecular clonal heterogeneity exists in esophageal squamous cell carcinomas. Therefore, care is necessary in preoperative genetic diagnosis using biopsy samples.

CPT‐11 May Provide Therapeutic Efficacy for Esophageal Squamous Cell Cancer and the Effects Correlate with the Level of DNA Topoisomerase I Protein

CPT‐11 is a potent anti‐cancer drug and a specific inhibitor of DNA topoisomerase I (Topo I). In this study, we aim to evaluate the effects of CPT‐11 on esophageal squamous cell cancers (ESCC) and to determine the correlation between the effects and the levels of Topo I expression. We examined the growth‐inhibitory effect caused by SN‐38, an active metabolite of CPT‐11, in 14 human ESCC cell lines established from 10 primary and 4 metastatic lesions. CPT‐11 was considered effective against 5 cell lines from primary lesions and one from metastatic lesions, and thus may show therapeutic efficacy against both primary and metastatic ESCC tumors. Although Topo I mRNA levels in these 14 ESCC cell lines, as quantitated by northern blot analysis, showed no correlation with the IC50 values, Topo I protein levels, as quantitated by western blot analysis, showed an inverse correlation with the IC50 values. Topo I protein levels could be an indicator of sensitivity to CPT‐11. We also determined Topo I protein levels in 40 ESCC tumors and matched normal mucosae. Thirty‐four tumors showed 1.2‐22.3‐fold increases in Topo I levels. Two patients receiving pre‐operative chemotherapy and one receiving radiotherapy exhibited increased Topo I protein levels in their tumor lesions. It appeared that CPT‐11 could provide selective therapeutic efficacy against ESCC tumors. CPT‐11 may be effective for the treatment of metastatic ESCC tumors and as a second‐line anti‐cancer drug for ESCC.

A survey of palliative medicine education in Japan’s undergraduate medical curriculum

BackgroundThis study aimed to examine the status of undergraduate palliative care education among Japanese medical students using data from a survey conducted in 2015.MethodsA questionnaire was originally developed, and the survey forms were sent to universities. The study’s objectives, methods, disclosure of results, and anonymity were explained to participating universities in writing. Responses returned by the universities were considered to indicate consent to participate. Descriptive statistical methodology was employed.ResultsThe response rate was 82.5% (66 of 80 medical faculties and colleges). Palliative care lectures were implemented in 98.5% of the institutions. Regarding lecture titles, “palliative medicine,” “palliative care,” and “terminal care” accounted for 42.4, 30.3, and 9.1% of the lectures, respectively. Teachers from the Department of Anesthesia, Palliative Care, and Psychiatry administered 51.5, 47.0, and 28.8% of lectures, respectively. Subjects of lectures included general palliative care (81.8%), pain management (87.9%), and symptom management (63.6%). Clinical clerkship on palliative care was a compulsory and non-compulsory course in 43.9 and 25.8% of the schools, respectively; 30.3% had no clinical clerkship curriculum.ConclusionsUndergraduate palliative care education is implemented in many Japanese universities. Clinical clerkship combined with participation in actual medical practice should be further improved by establishing a medical education certification system in compliance with the international standards.

Prognostic Factors for Post-Recurrence Survival in Patients with Thoracic Esophageal Squamous Cell Carcinoma after Curative Resection

Background/Aims: We investigated factors affecting the post-recurrence prognosis in order to develop adequate therapeutic guidelines for determining the indication for treatment of recurrent esophageal squamous cell carcinoma (ESCC). Methods: Ninety-three thoracic ESCC patients who developed postoperative recurrence after undergoing curative esophagectomy with 3-field lymphadenectomy at our institute and who received treatment for recurrence were enrolled in this study. Results: A univariate analysis showed that distant organ recurrence, the number of recurrent tumors, the longest diameter of the largest recurrent tumor, the sum of all of the longest diameters of the recurrent tumors, invasion into adjacent structures and the speed of growth of the representative recurrent tumors were significantly associated with the post-recurrence prognosis. A multivariate analysis showed that distant organ recurrence, the longest diameter of the largest recurrent tumor, invasion into adjacent structures and the speed of growth of the representative recurrent tumors were significantly associated with the prognosis. The patients whose recurrent tumor(s) did not meet all 4 of these factors showed a better prognosis. In contrast, the prognosis of the patients whose recurrent tumor(s) demonstrated all 4 factors was almost the same as that observed in the patients who did not receive any treatment for recurrence. Conclusions: The evaluation of these 4 factors may potentially be used to determine the indication for treatment for recurrence.