Kiyoko Yamamoto

Use of lectins as probes for analyzing embryonic induction

SummaryLectins were used as probes to investigate the mechanism of embryonic induction. Concanavalin (Con A) and gorse agglutinin out of 7 species of lectins tested were found to have strong neural-inducing effect on the presumptive ectoderm of newt gastrulae. Their effects were abolished by the addition of α-methyl-D-mannoside and α-L-fucose, respectively. Succinyl-Con A had a weak inducing activity in comparison to Con A. Autoradiography of3H-Con A-treated explants revealed that Con A bound to the inner surface, but not to the outer surface of ectoderm and was successively incorporated into cytoplasm.3H-Thymidine incorporation was lower in the first half and higher in the second half of the 60 h cultivation period in Con A-treated explants as compared to controls.Con A-Sepharose had a strong inductive effect. This suggests that neural induction is caused through Con A binding to the plasma membrane, but not through incorporation into the cytoplasm of the ectoderm cells.

Glycoproteins responsive to the neural-inducing effect of Concanavalin A in Cynops presumptive ectoderm

To examine the possible occurrence of receptors in the ectodermal cell surface which apparently mediates the neural-inducing stimulus, a further experiment by using Con A was done in combination with the enzyme treatments. The presumptive ectoderm explants of Cynops gastrula were first treated with neuraminidase to remove sialic acid. Prior to the Con A treatment, the explants were treated with almond glycopeptidase, which cleaves the asparagine linkage between protein and oligosaccharide in glycoprotein and releases the oligosaccharide moiety intact containing mannose residue from the substrate. No neural induction occurred. When the explants were not treated with almond glycopeptidase, the neural induction frequency was found to be the same as that of the explants treated with only Con A. Biochemical analyses showed that when the fixed ectoderm explants were treated with almond glycopeptidase, several oligosaccharides were released and then fractionated by means of Bio-Gel P-4 filtration. Based on the strict specificity of almond glycopeptidase, these oligosaccharides are unmistakably asparagine-linked oligosaccharides with mannose residues. We discuss the hypothesis of involvement of glycoproteins in the first step of molecular events in the neural induction mechanism.

Cell surface changes of the presumptive ectoderm following neural-inducing treatment by concanavalin A

SummaryScanning electron microscopic studies revealed that Concanavalin A (ConA) induces characteristic changes of the cell surface and the cell architecture of the presumptive ectoderm associated with differentiation into neural tissues. In Con A-treated cells, the filopodia with which cells were connected to each other disappeared from the interior (blastocoelic) surface and the cellular adhesivity decreased significantly. Thereafter, the cells underwent from those of the control explants. After cultivation for 60 h, a certain pattern of cell arrangement, which resembled the architecture of neural tissues, was observed among randomly arranged cells in the explants treated with Con A. The morphological changes specifically observed in Con A-treated explants were different from those found in explants treated with succinyl Con A (S-Con A) orDolichos biflorus agglutinin (DBA), which is unable to induce formation of the neural tissues. The molecular organization of the plasma membrane appears to be important in the mechanism of neural induction.

Clonal heterogeneity of mantle cell lymphoma revealed by array comparative genomic hybridization

Mantle cell lymphoma (MCL) is an aggressive B‐cell non‐Hodgkin lymphoma (NHL) characterized by the translocation t(11;14)(q13;q32). This lymphoma exhibits a poor prognosis and remains incurable with standard chemotherapy approaches. Recently, we have shown that a majority of patients with acute‐type adult T‐cell leukemia/lymphoma (ATLL) have multiple subclones that were likely produced in lymph nodes. We investigated whether MCL has multiple subclones as identified in ATLL by high‐resolution oligo‐array comparative genomic hybridization (CGH). Eleven of 20 (55%) evaluable MCL cases had a log2 ratio imbalance, suggesting the existence of multiple subclones in MCL. Based on the proportion of every subclone relative to the main clone, we were able to speculate clonal evolution in each MCL case with multiple subclones. Our analysis gave new insights into the clonal heterogeneity quantitatively and accurately. Furthermore, genomic copy number alterations are not hierarchical events and not necessarily the initial or later events for cells to become MCL.

CHANGES OF CHROMOSOMES DURING THE EARLY NEURAL DEVELOPMENT OF A JAPANESE NEWT, CYNOPS PYRRHOGASTER

The karyotype of Cynops pyrrhogaster was determined on the mitotic chromosomes in the presumptive neural area of an early gastrula. 24 chromosomes of a diploid set consisted of 8 metacentric and 4 submetacentric pairs. Individual chromosomes were identified on the basis of their morphology and characteristic C‐binding patterns. Sex chromosomes were not identified. Total length of the haploid chromosome set in the presumptive neural area decreased remarkably from morulae to gastrulae, further continued to decrease up to neurulae and thereafter remained unchanged till tail‐buds. Chromosome shortening occurring from morulae to gastrulae was accompanied with a prominent decrease in chromosome volume, keeping chromosome width constant. Shortening took place evenly along the longitudinal axis of a chromosome. When gastrulae and neurulae were compared concerning their positions of the appearance of the C‐bands, the basic pattern remained unchanged. In certain chromosomes, the number of C‐bands decreased as the result of their fusion, as gastrulae proceeded to neurulae.

A Molecular Aspect of Neural Induction in Cynops Presumptive Ectoderm Treated with Lectins

Little has been reported on what happens when the competent ectoderm first receives the inductive stimulus, or the location of the cellular site on which the inductive stimulus exerts its effect. Recently, the primary involvement of cell surface in the inducing mechanism of neural tissues has been stressed (Tiedemann and Born, 1978; Grunz and Staubach, 1979; Takata et al., 1981, 1984; Yamamoto et al., 1981; Duprat et al., 1982). Experiments using Sepharose beads, on which the neural-inducing factor (vegetalizing factor: Tiedemann and Born, 1978; Con A: Takata et al., 1981) was immobilized, indicated that even if the neural-inducing factor acts on the cell surface alone, it can evoke the competent ectoderm to differentiate into neural tissues, and does not necessarily have to enter into the cytoplasm to exert its effect. This possible mode of inducing action of Con A seems to agree with the evidence that retention of a certain amount of mitogen (Con A) for a certain length of time on the cell surface is a prerequisite for stimulating lymphocytes to blasto formation, but that mitogen taken up into cytoplasm is not involved in the mechanism of stimulation of cell division (Pauli et al., 1973).

Functionally deregulated AML1/RUNX1 cooperates with BCR-ABL to induce a blastic phase-like phenotype of chronic myelogenous leukemia in mice

Patients in the chronic phase (CP) of chronic myelogenous leukemia (CML) have been treated successfully following the advent of ABL kinase inhibitors, but once they progress to the blast crisis (BC) phase the prognosis becomes dismal. Although mechanisms underlying the progression are largely unknown, recent studies revealed the presence of alterations of key molecules for hematopoiesis, such as AML1/RUNX1. Our analysis of 13 BC cases revealed that three cases had AML1 mutations and the transcript levels of wild-type (wt.) AML1 were elevated in BC compared with CP. Functional analysis of representative AML1 mutants using mouse hematopoietic cells revealed the possible contribution of some, but not all, mutants for the BC-phenotype. Specifically, K83Q and R139G, but neither R80C nor D171N mutants, conferred upon BCR-ABL-expressing cells a growth advantage over BCR-ABL-alone control cells in cytokine-free culture, and the cells thus grown killed mice upon intravenous transfer. Unexpectedly, wt.AML1 behaved similarly to K83Q and R139G mutants. In a bone marrow transplantation assay, K83Q and wt.AML1s induced the emergence of blast-like cells. The overall findings suggest the roles of altered functions of AML1 imposed by some, but not all, mutants, and the elevated expression of wt.AML1 for the disease progression of CML.

Clinical value of flow cytometric immunophenotypic analysis for minimal residual disease detection in autologous stem-cell products of follicular and mantle cell lymphomas

Clinical value of flow cytometric immunophenotypic analysis for minimal residual disease detection in autologous stem-cell products of follicular and mantle cell lymphomas

Isolation of human mAbs that directly modulate FMS-related tyrosine kinase 3 signaling.

FMS‐related tyrosine kinase 3 (FLT3) is a class III receptor tyrosine kinase that plays important roles in hematopoiesis, including early progenitors and dendritic cell development. FLT3 is expressed at high levels in 70–100% of cases of AML and in virtually all cases of B‐lineage acute lymphoblastic leukemia. FLT3 is regarded as a molecular target in the development of novel therapies for acute leukemia patients. Currently, many small‐molecule FLT3 inhibitors have been developed, but clinical trials have resulted in limited antileukemia effects because of off‐target toxicities and drug resistance. The development of anti‐FLT3 Abs might overcome these difficulties and enhance the antileukemia efficacy of FLT3 inhibitors. In the present study, we demonstrate the isolation of novel human mAbs against FLT3 with antagonistic or agonistic activities. An antagonistic Ab, designated A2, continuously inhibits FLT3 ligand (FL)‐induced phosphorylation of FLT3 and MAPK. A2 cooperatively induces apoptosis with daunorubicin, even in the presence of FL. An agonistic Ab, designated 3E6, surprisingly induces the phosphorylation of FLT3 and MAPK, and supports the growth of a factor‐dependent cell line independently of FL addition. In addition, A2 showed complement‐dependent cytotoxicity activity, but was devoid of Ab‐dependent cell mediated cytotoxicity. Finally, we evaluated Ab internalization in a cell line. Immunofluorescence and flow cytometry analyses revealed that A2 is efficiently internalized. Collectively, these data demonstrate that A2 is a potent human Ab that might be capable of delivering cytotoxic reagents and that has antagonistic effects on FLT3 signaling. In addition, 3E6 might be a potential scaffold for novel dendritic cell‐based immunotherapies. (Cancer Sci 2012; 103: 350–359)

Rearrangement of VPS13B , a causative gene of Cohen syndrome, in a case of RUNX1 – RUNX1T1 leukemia with t(8;12;21)

Variant chromosomal translocations associated with t(8;21) are observed in 3–4% of acute myeloid leukemia (AML) cases with a RUNX1–RUNX1T1 fusion gene. However, the molecular events that occur in variants of t(8;21) are not well characterized. In the present study, we report genetic features of a variant three-way translocation of t(8;12;21)(q22;p11;q22) in a patient with AML. In this patient, leukemia cells lacked azurophilic granules, which does not correspond with the classic features of t(8;21). RNA-seq analysis revealed that TM7SF3 at 12p11 was fused to VPS13B at 8q22 and VPS13B to RUNX1, in addition to RUNX1–RUNX1T1. VPS13B was located near RUNX1T1 and both were localized at the same chromosomal bands. The reading frames of TM7SF3 and VPS13B did not match to those of VPS13B and RUNX1, respectively. Disruption of VPS13B causes Cohen syndrome, which presents intermittent neutropenia with a left-shifted granulopoiesis in the bone marrow. Disruption of VPS13B may thus cause the unusual features of RUNX1–RUNX1T1 leukemia. Our case indicates that rearrangement of VPS13B may be additional genetic events in variant t(8;21).