Toshio Watanabe

A Role for Hematopoietic Stem Cells in Promoting Angiogenesis

Angiogenesis is an important event for embryonic organogenesis as well as for tissue repair in the adult. Here, we show that hematopoietic stem cells (HSCs) play important roles for angiogenesis during embryogenesis. To investigate the role of HSCs in endothelial cell (EC) development, we analyzed AML1-deficient embryos, which lack definitive hematopoiesis. These embryos showed defective angiogenesis in the head and pericardium. Para-aortic splanchnopleural (P-Sp) explant cultures on stromal cells (P-Sp culture) did not generate definitive hematopoietic cells and showed defective angiogenesis in the AML1 null embryo. Disrupted angiogenesis in P-Sp cultures from AML1 null embryos was rescued by addition of HSCs or angiopoietin-1 (Ang1). HSCs, which express Ang1, directly promoted migration of ECs in vivo and in vitro. These results indicate that HSCs are critical for angiogenesis.

Follow-up results after resection of thoracic esophageal carcinoma

During the past 14.5 years, we have treated 430 patients with carcinoma of the thoracic esophagus. The resection rate was 72% and the overall operative mortality rate was 16%. During the past 5 years, the operative mortality rate has been markedly reduced to 4.5%. The low operative mortality rate in recent years is the result of selection of the operative procedure according to the criteria that we have established for one-stage radical resection. The overall 5-year survival rate was 16.5%. Prophylactic postoperative irradiation improved long-term survival in patients who underwent curative resection; their 5-year survival rate was 34.6%. Prophylactic irradiation was especially effective in patients who did not have lymph node metastases. The 5-year survival rate was 87.5% in these patients. The majority of long-term survivors were leading a normal life for their age.RésuméNous avons, au cours des 14.5 dernières années, traité 430 cancers de loesophage thoracique. Une résection a pu être réalisée dans 72% des cas. La mortalité opératoire globale est de 16%. Elle a été réduite à 4.5% pour les 5 dernières années, par une sélection du type dopération en fonction des critères que nous avons établis pour la résection en un temps. La survie globale à 5 ans est de 16.5%. Lirradiation prophylactique préopératoire améliore la survie chez les malades qui ont une résection à visée curatrice: leur survie à 5 ans est de 34.6%. Lirradiation prophylactique est particulièrement efficace chez les patients qui nont pas de métastases ganglionnaires: la survie à 5 ans est de 87.5%. La majorité des survivants mènent, pour leur áge, une vie normale.

Diminution of the AML1 Transcription Factor Function Causes Differential Effects on the Fates of CD4 and CD8 Single-Positive T Cells

In the thymic cortex, T lymphocytes are positively selected to survive and committed either to the CD4 single-positive (SP) or the CD8 SP lineage. The SP cells then pass through a step of maturation in the medulla and are delivered to peripheral lymphoid tissues. We examined the role of AML1, the gene encoding a transcription factor, in the above processes by using the transgenic mice expressing a dominant interfering form of AML1 as well as mice targeted heterozygously for AML1. One phenotypic change seen in the AML1-diminished mice was the reduction in the numbers of both CD4 SP and CD8 SP thymocytes, reflecting the partial impairment of the transition from the double-positive to SP stage. In addition, distinct from the above abnormality, perturbed were several aspects of SP cells, including the maturation of SP thymocytes, the recent thymic emigration, and the proliferative responsiveness of peripheral T cells to TCR stimulation. Interestingly, the AML1 diminution caused inhibitory and enhancing effects on the CD4 SP and CD8 SP cells, respectively. These differential effects are most likely related to the reduction in the peripheral CD4 SP/CD8 SP ratio observed in the AML1-diminished mice. The AML1 transcription factor thus maintains the homeostasis of each SP subset by functioning at the later stages of T lymphocyte differentiation.

Identification and functional characterization of paxillin as a target of protein tyrosine phosphatase receptor T

Protein tyrosine phosphatase receptor-type T (PTPRT) is the most frequently mutated tyrosine phosphatase in human cancers. However, the cell signaling pathways regulated by PTPRT largely remain to be elucidated. Here, we show that paxillin is a direct substrate of PTPRT and that PTPRT specifically regulates paxillin phosphorylation at tyrosine residue 88 (Y88) in colorectal cancer (CRC) cells. We engineered CRC cells homozygous for a paxillin Y88F knock-in mutant and found that these cells exhibit significantly reduced cell migration and impaired anchorage-independent growth, fail to form xenograft tumors in nude mice, and have decreased phosphorylation of p130CAS, SHP2, and AKT. PTPRT knockout mice that we generated exhibit increased levels of colonic paxillin phosphorylation at residue Y88 and are highly susceptible to carcinogen azoxymethane-induced colon tumor, providing critical in vivo evidence that PTPRT normally functions as a tumor suppressor. Moreover, similarly increased paxillin pY88 is also found as a common feature of human colon cancers. These studies reveal an important signaling pathway that plays a critical role in colorectal tumorigenesis.

Hemocytes of Ciona intestinalis express multiple genes involved in innate immune host defense.

Ascidians, which are classified as urochordata, appear to employ a primitive system of host defense that is considered to be a prototype of vertebrate innate immunity. We performed a cDNA/EST study to identify the genes expressed in the hemocytes of Ciona intestinalis. We obtained 3357 one-path reads that were then grouped into 1889 independent clusters. Although two thirds of the clusters could not be assigned to any particular gene, the remaining 530 clusters had significant homology to genes with known function. Of these, 62 clusters appeared to be related to host defense mechanisms. These include transcripts whose products are probably involved in cytotoxicity, detoxification, inflammation, and apoptosis. As expected, elements of acquired immunity were not detected. Thus, Ciona hemocytes appear to express a number of host defense-related genes involved in innate immune mechanisms.

Morpholino Antisense Oligonucleotide-Mediated Gene Knockdown During Thymocyte Development Reveals Role for Runx3 Transcription Factor in CD4 Silencing During Development of CD4−/CD8+ Thymocytes

During thymic T cell development, immature CD4+/CD8+ thymocytes develop into either CD4+/CD8− helper or CD4−/CD8+ CTLs. The molecular mechanisms governing the complex selection and differentiation steps during thymic T cell development are not well understood. Here we developed a novel approach to investigate gene function during thymocyte development. We transfected ex vivo isolated immature thymocytes with gene-specific morpholino antisense oligonucleotides and induced differentiation in cell or organ cultures. A morpholino oligonucleotide specific for CD8α strongly reduces CD8 expression. To our knowledge, this is the first demonstrated gene knockdown by morpholino oligonucleotides in primary lymphocytes. Using this approach, we show here that the transcription factor Runx3 is involved in silencing of CD4 expression during CD8 T cell differentiation. Runx3 protein expression appears late in thymocyte differentiation and is confined to mature CD8 single-positive thymocytes, whereas Runx3 mRNA is transcribed in mature CD4 and CD8 thymocytes. Therefore, Runx3 protein expression is regulated at a post-transcriptional level. The knockdown of Runx3 protein expression through morpholino oligonucleotides inhibited the development of CD4−/CD8+ T cells. Instead, mature cells with a CD4+/CD8+ phenotype accumulated. Potential Runx binding sites were identified in the CD4 gene silencer element, which are bound by Runx protein in EMSAs. Mutagenesis of potential Runx binding sites in the CD4 gene silencer abolished silencing activity in a reporter gene assay, indicating that Runx3 is involved in CD4 gene silencing. The experimental approach developed here should be valuable for the functional analysis of other candidate genes in T cell differentiation.

Hematopoietic cells in cultures of the murine embryonic aorta–gonad–mesonephros region are induced by c-Myb

Definitive hematopoiesis begins in the para-aortic, splanchnopleural (P-Sp) and aorta-gonad-mesonephros (AGM) regions of mouse embryos and then switches to the fetal liver [1] [2] [3]. Gene-targeted mice lacking the c-Myb transcription factor have severe hematopoietic defects in the fetal liver [4]. The role of c-Myb, if any, in P-Sp/AGM hematopoiesis has not been examined, however. Recently, we reported that oncostatin M can effectively expand both hematopoietic and endothelial-like cells from in vitro cultures of the AGM region [5]. Using this cell culture system, we examined the involvement of c-Myb in definitive hematopoiesis in the P-Sp and AGM regions. When primary cultures from the P-Sp or AGM regions of wild-type mouse embryos were probed with an anti-c-Myb antibody, hematopoietic cells but not endothelial-like cells showed positive staining. In contrast, in the P-Sp/AGM culture from c-myb(-/-) embryos, no hematopoietic cells were generated and endothelial-like cells predominated, indicating that the impairment of hematopoiesis in the liver of c-myb(-/-) embryos is actually preceded by a defect in P-Sp/AGM hematopoiesis. Hematogenic precursor cells were, however, still present in an inert but competent form among the endothelial-like, adherent cell population of c-myb(-/-) P-Sp/AGM cultures. When infected with a retrovirus carrying c-myb cDNA, these cultures gave rise to a significant number of hematopoietic cells. The rescued cells, unlike wild-type hematopoietic cells, were negative for c-Kit (a marker of hematopoietic progenitors), but did express other hematopoietic cell surface markers such as Mac-1, Gr-1 (myeloid markers), CD19, B220, Thy-1.2 (Iymphoid markers), and Ter119 (an erythroid marker). Thus, c-Myb plays a role in the generation of hematopoietic cells in the embryonic P-Sp and AGM regions.

Clathrin dependent endocytosis of E-cadherin is regulated by the Arf6GAP isoform SMAP1

E-cadherin is a central component of the adherens junction in epithelial cells and continuously undergoes endocytosis via clathrin-coated vesicles and/or caveolae depending on the cell type. In this study, we examined the role of SMAP1, a clathrin-interacting GTPase-activating protein (GAP) for the ADP-ribosylation factor 6 (Arf6) GTPase, in E-cadherin endocytosis. Mardin-Darby canine kidney (MDCK) epithelial cells were used as a model, and SMAP1 localized in the cytoplasm and along the adherens junction where E-cadherin was present. Next, activity of SMAP1 was compared with that of other Arf6GAPs (and/or an effector of Arf6-GTP), namely GIT1 and AMAP2/DDEF2. Overexpression of SMAP1 but not GIT1 nor AMAP2/DDEF2 strongly inhibited basal, as well as phorbolester-induced, internalization of E-cadherin. Notably, AMAP2/DDEF2 rather enhanced the caveolae-mediated incorporation of a membrane protein other than E-cadherin. Thus, in MDCK cells, E-cadherin appeared to be endocytosed solely through SMAP1-regulated clathrin-coated vesicles. Furthermore, MDCK cells overexpressing SMAP1 showed a reduced degree of cell migration compared to untransfected cells, as assessed by wound healing and Transwell assays, and this reduction in migration appeared to be due to the accumulation of E-cadherin at the adherens junction in cells overexpressing SMAP1. Collectively, SMAP1 likely represents a key Arf6GAP in clathrin dependent endocytosis of E-cadherin in MDCK cells. This activity of SMAP1 in E-cadherin turnover may be involved in epithelial organization and/or epithelial-mesenchymal transition.

Long-term evaluation of patients treated by radical operation for carcinoma of the thoracic esophagus

During the past 19 years, resection has been performed on 457 (72.9%) of 627 patients with carcinoma of the thoracic esophagus; curative resection was done in 388 patients. Among the 259 (57%) patients who underwent reconstruction through the retromediastinal space, thoracic esophagogastrostomy accounted for 181 and interposition of jejunal segments between the thoracic esophagus and the stomach accounted for 78 patients. Among the 171 (37.4%) patients who underwent reconstruction through the retrosternal space, there were 110 with esophagogastrostomy, 57 with interposition of jejunal segments, and 4 with colonic segments between the esophagus and the stomach. Comparing the reconstructive organs or the reconstructive routes used, there were no significant differences in the incidences of pulmonary complication, leakage, or operative death.Among the patients who tolerated curative surgery, the 5-year and 10-year survival rates were 31% and 23.1%, respectively. Comparing operative procedures, there were no significant differences in survival rates. Reconstruction was also performed in postgastrectomized patients using jejunal segments.There was a considerable number of complaints of passage failure and reflux esophagitis such as heartburn among patients who underwent esophagogastrostomy performed in the thoracic cavity, even 5 years after surgery.The performance status of patients in whom jejunal segments were used was better than that of patients in whom gastric tubes were used.RésuméAu cours des 19 dernières années, 457 résections (72.9%) ont été pratiquées chez 627 malades atteints de cancer de loesophage dont 388 opérations à visée curatrice. Deux cent cinquante neuf interventions (57%) ont été conduites par voie rétromédiastinale soit 181 oesophago-gastrostomies sans interposition et 78 avec interposition dune anse jéjunale après résection de la tumeur. Pendant la même période, cent soixante et onze (41%) ont été menées par voie rétro-sternale comprenant 110 oesophago-gastrostomies sans interposition dun segment intestinal, 57 avec interposition dun segment colique. La fréquence des complications pulmonaires, des fistules, des décès a été identique quelles quaient été la voie dabord empruntée et la méthode suivie pour rétablir la continuité digestive. Le taux de survie des opérés qui franchirent le cap de lintervention fut de 31% à 5 ans et 23.1% à 10 ans. Aucune différence du taux de survie en fonction des diverses opérations pratiquées na été enregistrée. En revanche, un grand nombre de troubles postopératoires tels que difficultés de transit du bol alimentaire et brûlures rétro-sternales dues à une oesophagite par reflux ont été notés quand la résection tumorale avait été éffectuée par voie intra-thoracique. De même il a été remarqué que létat des opérés était plus satisfaisant quand un segment de jéjunum était interposé entre loesophage et lestomac au lieu dun tube constitué à laide de lestomac.ResumenEn el curso de los últimos 19 años se ha realizado resección quirúrgica en 457 (72.9%) de 627 pacientes con carcinoma del esófago torácico; resección curativa fué posible en 388 casos. En 259 casos (57%) reconstruídos a través del espacio retromediastinal, la esofagogastrostomía torácica fué realizada en 181 casos y la interposición de segmentos yeyunales entre el esófago torácico y el estómago en 78 casos. Entre 171 casos (41%) reconstruídos a través del espacio retroesternal, hubo 110 casos de esofagogastrostomía, 57 de interposición de segmentos yeyunales y 4 de interposición de segmentos de colon entre el esófago y el estómago. Al comparar los órganos o la vía utilizados para la reconstrucción, no se hallaron diferencias significativas en la incidencia de complicaciones pulmonares, escapes anastomóticos o mortalidad operatoria.En los pacientes que toleraron le circugía curativa se encontraron tasas de supervivencia a 5 años y a 10 años de 31% y 23.1% respectivamente. Al comparar los procedimientos operatorios no se encuentran diferencias significativas en las tasas de supervivencia. La reconstrucción también fué realizada en pacientes postgastrectomizados utilizando segmentos de yeyuno.Hubo un número considerable de quejas de fallas en deglución y esofagitis de reflujo, tales como pirosis, en los pacientes sometidos a esofagogastrostomía realizada en la cavidad torácica, aún 5 años después de la circugía. El estado funcional de los pacientes en quienes se utilizaron segmentos yeyunales fué mejor que el de los pacientes en quienes se utilizaron tubos gástricos.

Transplantation of germ nuclei in Paramecium caudatum: III. Role of germinal micronucleus in vegetative growth

Abstract By using the technique of nuclear transplantation in Paramecium [1], amicronucleate and renucleate clones were prepared in P. caudatum . The major differences between amicronucleate and micronucleate cells in the vegetative stage are elongation of cell cycle time, decrease in food vacuole formation, and shortening of the buccal cavity in the amicronucleate cells. These characteristics of amicronucleate cells are closely related with the absence of micronucleus, because all of these abnormalities were cured when the micronucleus was transplanted again into the amicronucleate. It is evident that the germinal micronucleus plays an important role not only during the sexual cycle but also in vegetative growth. Elongation of the cell cycle time in amicronucleates was also observed in P. bursaria and P. jenningsi .